CN109371018A - For dissociate Circulating tumor DNA extraction reinforcing agent, extract peripheral blood in dissociate Circulating tumor DNA kit and method - Google Patents
For dissociate Circulating tumor DNA extraction reinforcing agent, extract peripheral blood in dissociate Circulating tumor DNA kit and method Download PDFInfo
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- CN109371018A CN109371018A CN201811645358.4A CN201811645358A CN109371018A CN 109371018 A CN109371018 A CN 109371018A CN 201811645358 A CN201811645358 A CN 201811645358A CN 109371018 A CN109371018 A CN 109371018A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
The invention discloses the kits and method of the Circulating tumor DNA that dissociates in a kind of reinforcing agent extracted for the Circulating tumor DNA that dissociates, extraction peripheral blood.Wherein, which includes: the 1 μ g/ μ μ g/ μ L of L~5 Carrier RNA, the 1 μ g/ μ μ g/ μ L of L~5 polyadenylic acid, the 1 μ g/ μ μ g/ μ L of L~5 glycogen, 0~5 μ g/ μ L linear acrylamide, the 1 μ g/ μ μ g/ μ L yeast of L~5 tRNA, the 2 μ g/ μ μ of L~10 g/ μ L dog salmon sperm DNAs and/or herring sperm dna and water.Reinforcing agent for Circulating tumor DNA (ctDNA) extraction that dissociates of the invention, it liquid, washing buffer, eluent etc. can be used in combination in conjunction with conventional cracking, it by specific combination ctDNA and settles down, improves the extraction efficiency and extraction yield of ctDNA significantly.
Description
Technical field
The present invention relates to field of biomedicine technology, in particular to a kind of for the Circulating tumor DNA extraction that dissociates
Reinforcing agent, the kit and method for extracting the Circulating tumor DNA that dissociates in peripheral blood.
Background technique
With being continuously increased for morbidity and mortality, cancer has become the first cause of Chinese population death and main
Public health problem.Anti-cancer therapies are varied, including radiotherapy, chemotherapy and emerging targeted therapy and immune control
It treats；Wherein, targeted therapy is the important composition of accurate medicine, and particularly important for the screening of target gene specificity crowd,
The genetic test of cancer is directly related to drug for the therapeutic efficiency of patient.Currently, the main sample type for detection is
Tumor tissues puncture sample；But tissue samples are invasive operation, are difficult to carry out sometimes, and these operations are big by tumour
Small, position, patient's ordinary circumstance etc. influence, and cannot obtain satisfied tissue sometimes, there are a series of limitations.Tumour cell
Apoptosis release ctDNA enters peripheral blood after necrosis and the tumour cell to fall off enter blood.Plasma free Tumour DNA in recent years
It (ctDNA) is considered as a kind of sample that detectable tomour specific sexually revises.
CtDNA is for Tumor mutations detection advantage: operation is noninvasive or minimally invasive；It can all be obtained in any process of disease
It takes；It can be used as a kind of tumor marker, realize real-time detection and dynamic detection；Overcome the heterogeneity of tumor tissues.However, mesh
Preceding still to have some technical challenges using ctDNA detection gene mutation, be mainly shown as: 1) ctDNA content varies with each individual, and
And most people content is lower；2) ctDNA segment is relatively small, and most of is about 180bp, and segment is distributed in 100bp~400bp；
3) the relevant DNA proportion different people difference of tumour is larger in ctDNA, and often is difficult to measure because ratio is small.These limits
Extensive use of the ctDNA in lesion detection is made, therefore, the extraction of efficient easily ctDNA is to influence ctDNA in tumour
An important factor for being widely applied in detection.
Currently, the ctDNA extracts kit of commercialization mainly has column formulation and paramagnetic particle method, column formulation extraction efficiency is high, but
Column formulation is larger by sample matrix interference, needs the complex instruments such as centrifuge, vacuum pump；Paramagnetic particle method extraction is simple and fast, is not required to
Complex instrument is wanted, and manual operations, but the magnetic bead extracts kit being commercialized at present can be reduced in conjunction with work station etc. is extracted
Extraction efficiency is general, it would be highly desirable to solve the problems, such as extraction efficiency.
Summary of the invention
It is followed the present invention is intended to provide dissociating in a kind of reinforcing agent extracted for the Circulating tumor DNA that dissociates, extraction peripheral blood
The kit and method of ring Tumour DNA, to improve the extraction efficiency of free Circulating tumor DNA.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of mention for the Circulating tumor DNA that dissociates
The reinforcing agent taken.The reinforcing agent includes: the 1 μ g/ μ μ g/ μ of L~5 LCarrier RNA, the 1 μ g/ μ μ g/ μ L of L~5 polyadenylic acid, 1 μ
The g/ μ μ g/ μ L of L~5 glycogen, 0~5 μ g/ μ L linear acrylamide, the 1 μ g/ μ μ g/ μ L yeast of L~5 tRNA, the 2 μ g/ μ μ g/ μ of L~10 L
Dog salmon sperm DNA and/or herring sperm dna and water.
Further, the residue number of polyadenylic acid is 2100~10000nt, and the weight average molecular weight of linear acrylamide is
1000000~5,000,000.
Further, Carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, dog salmon essence
The mass ratio of sub- DNA and herring sperm dna is 1:1:1:1:1:1:1.
According to another aspect of the present invention, a kind of kit for extracting the Circulating tumor DNA that dissociates in peripheral blood is provided.It should
Kit includes any of the above-described kind of reinforcing agent for the Circulating tumor DNA extraction that dissociates.
Further, kit further includes bead suspension, SDS solution, cracking in conjunction with liquid, Proteinase K, protease dissolution
Buffer, the first cleaning solution, the second cleaning solution and eluent.
Further, it includes 45mmol/L Tris-HCl, 120mmol/L NaCl, 30mmol/L second two that cracking, which combines liquid,
Amine tetraacethyl disodium, 10~30mol/L guanidinium isothiocyanate, 2~4mol/L potassium acetate and the polysorbas20 of 5wt~10%, pH value are
7.6；Preferably, SDS solution concentration is 10%.
Further, protease dissolution buffer includes 45~75mmol/L Tris-HCl and 100~120mmol/
LNaCl。
Further, the first cleaning solution include 45~75mmol/L Tris-HCl, 100~120mmol/LNaCl, 30~
60mmol/L disodium ethylene diamine tetraacetate and 1.5wt% Qula are logical, pH value 8.0；Preferably, the second cleaning solution includes 80%
Ethyl alcohol；Preferably, eluent is nuclease-free water.
In accordance with a further aspect of the present invention, a kind of method for extracting the Circulating tumor DNA that dissociates in peripheral blood is provided.The party
Method extracts the Circulating tumor DNA that dissociates in peripheral blood using any of the above-described kind of kit.
Further, comprising the following steps: the following steps are included: 1mg Proteinase K 1) is previously dissolved in 500 in advance~
Proteinase K Solution is formed in 1000 μ L protease dissolution buffer, 15 μ L Proteinase K Solutions and 50 μ L are added into centrifuge tube
SDS solution；2) 1mL serum or plasma sample are shifted to containing in 15 μ L egg Proteinase K Solutions and 50 μ L egg SDS solution centrifuge tubes；
3) 60 DEG C of incubation 10min in water-bath are placed in, is vortexed and mixes 1 time every 5min；5min is placed on ice；4) it is outstanding that 15 μ L magnetic beads are added
Supernatant liquid, 1250 μ L cracking combine liquid into centrifuge tube, are vortexed and mix 30 seconds, add 2 μ L reinforcing agents, room temperature is mixed by inversion 15 points
Clock；5) it is transferred on magnetic frame, stands 3 minutes absorption magnetic beads, inhale and abandon solution；6) 500 the first cleaning solutions of μ L are added, is vortexed and mixes
30 seconds；7) it is transferred on magnetic frame, stands 3 minutes absorption magnetic beads, inhale and abandon solution；8) 500 the second cleaning solutions of μ L are added, are vortexed mixed
Even 30 seconds；9) it is transferred on magnetic frame, stands 3 minutes absorption magnetic beads, inhale and abandon solution；10) step 8) and 9) primary is repeated；11)
Centrifugation, collecting pipe are centrifuged the drop on tube wall, are transferred on magnetic frame, inhale and abandon solution；12) it is air-dried 3~5 minutes；13) add
The eluent of 52 μ L, vortex break up magnetic bead, are placed at room temperature for 5~10 minutes, during which shake 2~3 acceleration DNA dissolutions；13) it shifts
To magnetic frame, 3 minutes are stood, the DNA for shifting dissolution is saved into new centrifuge tube, and the DNA of dissolution is that free circulation is swollen
Tumor DNA.
The reinforcing agent for Circulating tumor DNA (ctDNA) extraction that dissociates of the invention, can in conjunction with conventional cracking liquid,
Washing buffer, eluent etc. are used in combination, and by specific combination ctDNA and settle down, improve ctDNA significantly
Extraction efficiency and extraction yield, solve current tumor peripheries blood genetic test clinically ctDNA extraction often extract
The problem of amount is insufficient, extracts failure, develops the potentiality of ctDNA detection, and that has widened clinical tumor detection ctDNA applies model
It encloses, improves the application value of ctDNA.
Detailed description of the invention
The accompanying drawings constituting a part of this application is used to provide further understanding of the present invention, and of the invention shows
Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.In the accompanying drawings:
Fig. 1 and Fig. 2 respectively illustrates the detection of the kit and DNA extraction kit A29319 clinical sample 1 of embodiment 1
As a result；
Fig. 3 and Fig. 4 respectively illustrates the detection of the kit and DNA extraction kit A29319 clinical sample 2 of embodiment 1
As a result；And
Fig. 5 and Fig. 6 respectively illustrates the detection of the kit and DNA extraction kit A29319 clinical sample 3 of embodiment 1
As a result.
Specific embodiment
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.The present invention will be described in detail below with reference to the accompanying drawings and embodiments.
It is a kind of according to the present invention that typically embodiment there is provided a kind of enhancings for the Circulating tumor DNA extraction that dissociates
Agent.The reinforcing agent includes: the 1 μ g/ μ μ g/ μ of L~5 LCarrier RNA, the 1 μ g/ μ μ g/ μ L of L~5 polyadenylic acid, 1 μ g/ μ L~5 μ
G/ μ L glycogen, 0~5 μ g/ μ L linear acrylamide, the 1 μ g/ μ μ g/ μ L yeast of L~5 tRNA, the 2 μ g/ μ μ g/ μ L dog salmons of L~10
Sperm DNA and/or herring sperm dna and water.
The reinforcing agent for Circulating tumor DNA (ctDNA) extraction that dissociates of the invention, can in conjunction with conventional cracking liquid,
Washing buffer, eluent etc. are used in combination, and by specific combination ctDNA and settle down, improve ctDNA significantly
Extraction efficiency and extraction yield, solve current tumor peripheries blood genetic test clinically ctDNA extraction often extract
The problem of amount is insufficient, extracts failure, develops the potentiality of ctDNA detection, and that has widened clinical tumor detection ctDNA applies model
It encloses, improves the application value of ctDNA.
Currently, commercialized settling agent selects single component to play a role alone mostly, and different ingredient advantages is different:
Dog salmon sperm DNA or herring sperm dna are difficult to be enriched with 60bp DNA fragmentation below, and polyadenylic acid but has significantly
Help heavy performance；The DNA fragmentation of 110bp or so, glycogen help heavy ability poor, and linear acrylamide's helps heavy performance better than glycogen.
Therefore, one-component has defect, or even occurs reaction in extraction efficiency, so that extraction efficiency reduces；The invention
The a variety of settling agent ingredients of selection of property, a variety of settling agents, which mutually cooperate with to play a role, can achieve mutual supplement with each other's advantages, supply a gap.Mirror
Larger (100bp~400bp) is fluctuated in ctDNA molecule fragment, therefore, a variety of settling agents play help heavy effect, Ke Yigeng simultaneously
High yield, more comprehensively, more completely obtain blood plasma in ctDNA.
Preferably, the residue number of polyadenylic acid be 2100~10000nt, more preferably 2100~5000nt, linearly
The weight average molecular weight of acrylamide is 1,000,000~5,000,000.The polyadenylic acid of this residue number and linear the third of this weight average molecular weight
Acrylamide is used cooperatively, can preferably isolated fragment size 100~400bp ctDNA.
It is furthermore preferred that Carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, dog salmon essence
The mass ratio of sub- DNA and herring sperm dna is 1:1:1:1:1:1:1.
A kind of typical embodiment according to the present invention provides a kind of examination for extracting the Circulating tumor DNA that dissociates in peripheral blood
Agent box.The kit includes any of the above-described kind of reinforcing agent for the Circulating tumor DNA extraction that dissociates.
The reinforcing agent for Circulating tumor DNA (ctDNA) extraction that dissociates of the invention, can in conjunction with conventional cracking liquid,
Washing buffer, eluent etc. are used in combination, and by specific combination ctDNA and settle down, improve ctDNA significantly
Extraction efficiency and extraction yield, solve current tumor peripheries blood genetic test clinically ctDNA extraction often extract
The problem of amount is insufficient, extracts failure, develops the potentiality of ctDNA detection, and that has widened clinical tumor detection ctDNA applies model
It encloses, improves the application value of ctDNA.
Preferably, for ease of use, kit further includes bead suspension, SDS solution, cracking in conjunction with liquid, protease
K, protease dissolution buffer, the first cleaning solution, the second cleaning solution and eluent.
It is furthermore preferred that it includes 45mmol/L Tris-HCl, 120mmol/LNaCl, 30mmol/L ethylenediamine that cracking, which combines liquid,
Tetraacethyl disodium, 10~30mol/L guanidinium isothiocyanate, 2~4mol/L potassium acetate and the polysorbas20 of 5wt~10%, pH value 7.6.
The cracking combination liquid can give full play to cracking function, have the advantages that cracking is thorough；Preferably, SDS solution concentration is 10%.
It is furthermore preferred that protease dissolution buffer includes 45~75mmol/L Tris-HCl and 100~120mmol/L
NaCl.The protease, which dissolves buffer, has dissolved efficiency high, to the not damaged advantage of DNA.
It is furthermore preferred that the first cleaning solution include 45~75mmol/L Tris-HCl, 100~120mmol/LNaCl, 30~
60mmol/L disodium ethylene diamine tetraacetate and 1.5wt% Qula are logical, pH value 8.0；First cleaning solution has detersive efficiency high
The advantages of.Preferably, the second cleaning solution includes 80% ethyl alcohol；Preferably, eluent is nuclease-free water.
A kind of typical embodiment according to the present invention provides a kind of side for extracting the Circulating tumor DNA that dissociates in peripheral blood
Method.This method extracts the Circulating tumor DNA that dissociates in peripheral blood using any of the above-described kind of kit.The kit can be significant
The extraction efficiency and extraction yield of ctDNA are provided.
Preferably, comprising the following steps: the following steps are included: 1mg Proteinase K 1) is previously dissolved in 500~1000 μ in advance
Proteinase K Solution is formed in L protease dissolution buffer, 15 μ L Proteinase K Solutions are added into centrifuge tube and 50 μ L SDS are molten
Liquid；2) 1mL serum or plasma sample are shifted to containing in 15 μ L egg Proteinase K Solutions and 50 μ L egg SDS solution centrifuge tubes；3) it is placed in
60 DEG C of incubation 10min in water-bath are vortexed every 5min and mix 1 time；5min is placed on ice；4) be added 15 μ L bead suspensions,
1250 μ L cracking combines liquid into centrifuge tube, is vortexed and mixes 30 seconds, adds 2 μ L reinforcing agents, room temperature is mixed by inversion 15 minutes；5)
It is transferred on magnetic frame, stands 3 minutes absorption magnetic beads, inhale and abandon solution；6) 500 the first cleaning solutions of μ L are added, is vortexed and mixes 30 seconds；
7) it is transferred on magnetic frame, stands 3 minutes absorption magnetic beads, inhale and abandon solution；8) 500 the second cleaning solutions of μ L are added, is vortexed and mixes 30
Second；9) it is transferred on magnetic frame, stands 3 minutes absorption magnetic beads, inhale and abandon solution；10) step 8) and 9) primary is repeated；11) it is centrifuged,
Collecting pipe is centrifuged the drop on tube wall, is transferred on magnetic frame, inhales and abandons solution；12) it is air-dried 3~5 minutes；13) add 52 μ L
Eluent, vortex breaks up magnetic bead, is placed at room temperature for 5~10 minutes, during which shake 2~3 accelerations DNA and dissolve；13) it is transferred to magnetic
On power frame, 3 minutes are stood, the DNA for shifting dissolution is saved into new centrifuge tube, and the DNA of dissolution is free circulating tumor
DNA。
In an embodiment of the present invention, kit is made of following reagent:
1. bead suspension G is purchased from life company；
2.SDS solution is purchased from life company, concentration 10%；
3. cracking combines liquid: 45mmol/L Tris-HCl, 120mmol/LNaCl, 30mmol/L ethylenediamine tetra-acetic acid two
Sodium, 10mol/L guanidinium isothiocyanate, 2mol/L potassium acetate, 5wt% polysorbas20, pH value 7.6；
4. Proteinase K is purchased from life company；
5. protease dissolves buffer: 45mmol/L Tris-HCl, 120mmol/LNaCl；
6. the first cleaning solution: 45mmol/L Tris-HCl, 120mmol/LNaCl, 30mmol/L ethylenediamine tetra-acetic acid two
Sodium, 1.5wt% Qula are logical, pH value 8.0；
7. the second cleaning solution: 80% ethyl alcohol；
8. eluent AE: nuclease-free water；
9. reinforcing agent: the 1 μ g/ μ μ g/ μ of L~5 LCarrier RNA, the 1 μ g/ μ μ g/ μ L of L~5 polyadenylic acid, 1 L~5 μ g/ μ
μ g/ μ L glycogen, 0~5 μ g/ μ L linear acrylamide, the 1 μ g/ μ μ g/ μ L yeast of L~5 tRNA, the big horse of the 2 μ g/ μ μ g/ μ of L~10 L breathe out
Fish sperm DNA and/or herring sperm dna and water, Carrier RNA, polyadenylic acid, glycogen, linear acrylamide (linear
Acrylamide), yeast tRNA, dog salmon sperm DNA, herring sperm dna, it is mixed by the mass ratio of 1:1:1:1:1:1:1
It closes buffer to be made, buffer is water.
Using the kit extract free ctDNA extracting method the following steps are included:
1. 15 μ L Proteinase Ks are added into the centrifuge tube of 2.0mL, and (1mg Proteinase K is previously dissolved in protease dissolution buffer
In) and 50 μ L SDS solution；
2. shifting 1mL serum or plasma sample into the centrifuge tube for containing 15 μ L Proteinase Ks and 50 μ L SDS solution；
3. being placed in 60 DEG C of incubation 10min in water-bath, it is vortexed and mixes 1 time every 5min；5min is placed on ice.
4. 15 μ L bead suspension G, 1250 μ L cracking of addition combines liquid into sample, it is vortexed and mixes 30 seconds, at this point, adding again
Enter 2 μ L enhancement solutions, room temperature is mixed by inversion 15 minutes；
5. being transferred on magnetic frame, 3 minutes absorption magnetic beads are stood, inhales and abandons all solution；
6. 500 μ L cleaning solutions 1 are added, it is vortexed and mixes 30 seconds；
7. being transferred on magnetic frame, 3 minutes absorption magnetic beads are stood, careful inhale abandons all solution；
8. 500 μ L cleaning solutions 2 are added, it is vortexed and mixes 30 seconds；
9. being transferred on magnetic frame, 3 minutes absorption magnetic beads are stood, careful inhale abandons all solution；
10. it is primary to repeat 7-8 step；
11. of short duration centrifugation is collected the drop on tube wall, is transferred on magnetic frame, careful inhale abandons all solution；
12. being air-dried 3-5 minutes；
13. adding the eluent AE of 52 μ L, vortex breaks up magnetic bead, is placed at room temperature for 5-10 minutes, during which gently shakes 2-3 times and adds
Fast DNA dissolution；
14. be transferred on magnetic frame, 3 minutes are stood, transfer DNA is into new 1.5ml centrifuge tube, and in condition appropriate
It saves.
Beneficial effects of the present invention are further illustrated below in conjunction with embodiment.The reagent do not write exactly in following example or
The reagent of routine of the invention can be used in method or technological means is realized.
Embodiment 1
The present embodiment kit is made of following reagent:
1. bead suspension G is purchased from life company；
2.SDS solution is purchased from life company, concentration 10%；
3. cracking combines liquid: 45mmol/L Tris-HCl, 120mmol/LNaCl, 30mmol/L ethylenediamine tetra-acetic acid two
Sodium, 10mol/L guanidinium isothiocyanate, 2mol/L potassium acetate, 5wt% polysorbas20, pH value 7.6；
4. Proteinase K is purchased from life company；
5. protease dissolves buffer: 45mmol/L Tris-HCl, 120mmol/LNaCl；
6. cleaning solution 1:45mmol/L Tris-HCl, 120mmol/LNaCl, 30mmol/L disodium ethylene diamine tetraacetate,
1.5wt% Qula is logical, pH value 8.0；
7. the ethyl alcohol of cleaning solution 2:80%；
8. eluent AE: nuclease-free water；
9. reinforcing agent: final concentration of 2.5 μ g/ μ LCarrier RNA, 2.5 μ g/ μ L polyadenylic acids, 2.5 μ g/ μ L glycogens,
2.5 μ g/ μ L linear acrylamides (linear acrylamide), 2.5 μ g/ μ L yeast tRNA, 2.5 μ g/ μ L dog salmon sperms
DNA, 2.5 μ g/ μ L herring sperm dnas, buffer is water.
The present embodiment dissociates ctDNA extracting method:
1. 15 μ L Proteinase Ks are added into the centrifuge tube of 2.0mL, and (1mg Proteinase K is previously dissolved in protease dissolution buffer
In) and 50 μ L SDS solution；
2. shifting 1mL serum or plasma sample into the centrifuge tube for containing 15 μ L Proteinase K Solutions and 50 μ L SDS solution；
3. being placed in 60 DEG C of incubation 10min in water-bath, it is vortexed and mixes 1 time every 5min；5min is placed on ice.
3. 15 μ L bead suspension G, 1250 μ L cracking of addition combines liquid into sample, it is vortexed and mixes 30 seconds, at this point, adding again
Enter 2 μ L enhancement solutions, room temperature is mixed by inversion 15 minutes；
4. being transferred on magnetic frame, 3 minutes absorption magnetic beads are stood, inhales and abandons all solution；
5. 500 μ L cleaning solutions 1 are added, it is vortexed and mixes 30 seconds；
6. being transferred on magnetic frame, 3 minutes absorption magnetic beads are stood, careful inhale abandons all solution；
7. 500 μ L cleaning solutions 2 are added, it is vortexed and mixes 30 seconds；
8. being transferred on magnetic frame, 3 minutes absorption magnetic beads are stood, careful inhale abandons all solution；
9. it is primary to repeat 7-8 step；
10. of short duration centrifugation is collected the drop on tube wall, is transferred on magnetic frame, careful inhale abandons all solution；
11. being air-dried 3-5 minutes；
12. adding the eluent AE of 52 μ L, vortex breaks up magnetic bead, is placed at room temperature for 5-10 minutes, during which gently shakes 2-3 times and adds
Fast DNA dissolution；
13. be transferred on magnetic frame, 3 minutes are stood, transfer DNA is into new 1.5ml centrifuge tube, and in condition appropriate
It saves.
One, the optimization of the present embodiment reinforcing agent heterogeneity ratio
Using the kit and method of the present embodiment, the optimization of reinforcing agent composition quality ratio is carried out, other than reinforcing agent,
Other components do not adjust, and are divided into three groups.1 group of reinforcing agent, Carrier RNA, polyadenylic acid, glycogen, linear acrylamide, ferment
Female tRNA, dog salmon sperm DNA and herring sperm dna example 1:1:1:1:1:1:1 in mass ratio mixing；Final concentration is 2.5 μ
g/μL.2 groups of reinforcing agent, Carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, dog salmon sperm
DNA and herring sperm dna example 1:2:1:2:1:2:1 in mass ratio mixing, final concentration is respectively 2.5 μ g/ μ L, 5 μ g/ μ L, 2.5 μ
G/ μ L, 5 μ g/ μ L, 2.5 μ g/ μ L, 5 μ g/ μ L, 2.5 μ g/ μ L.3 groups of reinforcing agent, Carrier RNA, polyadenylic acid, glycogen, line
Property acrylamide, yeast tRNA, dog salmon sperm DNA and herring sperm dna example 2:1:2:1:2:1:2 in mass ratio mixing；
Final concentration is respectively 5 μ g/ μ L, 2.5 μ g/ μ L, 5 μ g/ μ L, 2.5 μ g/ μ L, 5 μ g/ μ L, 2.5 μ g/ μ L, 5 μ g/ μ L.Extract 3 8ml
Clinical peripheral blood plasma sample；Each experiment flow 1ml is extracted, and each sample extracts 8 times altogether, after the DNA mixing of 8 extractions,
With qPCR quantitative measurment extracted amount, 1 as a result see the table below:
Table 1
Conclusion: 1 group of reinforcing agent, Carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, big horse
Breathe out fish sperm DNA and herring sperm dna example 1:1:1:1:1:1:1 in mass ratio mixing；Final concentration is 2.5 μ g/ μ L.Most substantially
Degree improves the extracted amount of ctDNA, realizes optimal ctDNA and helps heavy effect.
Two, using the kit of the present embodiment and method and life company paramagnetic particle method dissociative DNA extracts kit A29319
(abbreviation DNA extraction kit A29319) extracts 3 8ml clinic peripheral blood plasma samples；Each experiment flow 1ml is extracted, often
A sample extracts 8 times altogether, after the DNA mixing of 8 extractions, with qPCR quantitative measurment extracted amount, as a result see the table below 2:
Table 2
By 2 result of table as it can be seen that the ctDNA total amount that kit of the invention extracts is significantly higher than DNA extraction kit
A29319。
Three, clip size is measured with Tapestation 2100, respectively takes 2100 clip size of 2ng sample detection, this implementation
By FIG. 1 to FIG. 6, (wherein Fig. 1 and Fig. 2 is respectively the reagent of the present embodiment to the ctDNA that example is extracted with DNA extraction kit A29319
The testing result of box and DNA extraction kit A29319 clinical sample 1；Wherein Fig. 3 and Fig. 4 is respectively the kit of the present embodiment
With the testing result of DNA extraction kit A29319 clinical sample 2；Wherein Fig. 5 and Fig. 6 be respectively the present embodiment kit and
The testing result of DNA extraction kit A29319 clinical sample 3) as it can be seen that main sections size between 100bp~400bp,
It is in the same size with ctDNA main sections reported in the literature, show the present embodiment extracts kit excellent performance, extraction process pair
CtDNA is not damaged.
Four, each clinical sample of said extracted respectively take 20ng to execute identical library construction process, machine process on chip
With analysis of biological information process, compare testing result: clinical sample 2, our company's product extract testing result and clinical hospitals knot
Fruit is unanimously positive, and compares Products result as feminine gender.Show that I extracts excellent performance by Products, to extraction
CtDNA is not damaged.Detailed results see the table below 3:
Table 3
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of reinforcing agent for the Circulating tumor DNA extraction that dissociates characterized by comprising the 1 μ g/ μ μ g/ of L~5 μ
LCarrierRNA, the 1 μ g/ μ μ g/ μ L of L~5 polyadenylic acid, the 1 μ g/ μ μ g/ μ L of L~5 glycogen, 0~5 μ g/ μ L linear propylene's acyl
Amine, the 1 μ g/ μ μ g/ μ L yeast of L~5 tRNA, the 2 μ g/ μ μ of L~10 g/ μ L dog salmon sperm DNAs and/or herring sperm dna and water.
2. reinforcing agent according to claim 1, which is characterized in that the residue number of the polyadenylic acid be 2100~
10000nt, the weight average molecular weight of the linear acrylamide are 1,000,000~5,000,000.
3. reinforcing agent according to claim 1, which is characterized in that Carrier RNA, the polyadenylic acid, institute
State the matter of glycogen, the linear acrylamide, the yeast tRNA, the dog salmon sperm DNA and the herring sperm dna
Amount ratio is 1:1:1:1:1:1:1.
4. a kind of kit for extracting the Circulating tumor DNA that dissociates in peripheral blood, which is characterized in that including in such as claims 1 to 3
Described in any item reinforcing agents for the Circulating tumor DNA extraction that dissociates.
5. kit according to claim 4, which is characterized in that the kit further includes bead suspension, SDS molten
Liquid, cracking combine liquid, Proteinase K, protease dissolution buffer, the first cleaning solution, the second cleaning solution and eluent.
6. kit according to claim 5, which is characterized in that it includes 45mmol/L Tris- that the cracking, which combines liquid,
HCl, 120mmol/LNaCl, 30mmol/L disodium ethylene diamine tetraacetate, 10~30mol/L guanidinium isothiocyanate, 2~4mol/L vinegar
Sour potassium and the polysorbas20 of 5wt~10%, pH value 7.6；
Preferably, the SDS solution concentration is 10%.
7. kit according to claim 5, which is characterized in that protease dissolution buffer includes 45~
75mmol/LTris-HCl and 100~120mmol/LNaCl.
8. kit according to claim 5, which is characterized in that first cleaning solution includes 45~75mmol/L
Tris-HCl, 100~120mmol/LNaCl, 30~60mmol/L disodium ethylene diamine tetraacetate and 1.5wt% Qula are logical, pH value
It is 8.0；
Preferably, second cleaning solution includes 80% ethyl alcohol；
Preferably, the eluent is nuclease-free water.
9. a kind of method for extracting the Circulating tumor DNA that dissociates in peripheral blood, which is characterized in that using as appointed in claim 4 to 8
Kit described in one extracts the Circulating tumor DNA that dissociates in peripheral blood.
10. according to the method described in claim 9, characterized by comprising the following steps:
1) 1mg Proteinase K is previously dissolved in 500~1000 μ L protease dissolution buffer in advance and forms Proteinase K Solution, to
Proteinase K Solution described in 15 μ L and 50 μ LSDS solution are added in centrifuge tube；
2) shift 1mL serum or plasma sample to contain described in egg SDS solution described in egg Proteinase K Solution described in 15 μ L and 50 μ L from
In heart pipe；
3) 60 DEG C of incubation 10min in water-bath are placed in, is vortexed and mixes 1 time every 5min；5min is placed on ice；
4) 15 μ L bead suspensions, 1250 μ L cracking is added, into the centrifuge tube, to be vortexed and mix 30 seconds, add 2 μ in conjunction with liquid
L reinforcing agent, room temperature are mixed by inversion 15 minutes；
5) it is transferred on magnetic frame, stands 3 minutes absorption magnetic beads, inhale and abandon solution；
6) 500 the first cleaning solutions of μ L are added, is vortexed and mixes 30 seconds；
7) it is transferred on magnetic frame, stands 3 minutes absorption magnetic beads, inhale and abandon solution；
8) 500 the second cleaning solutions of μ L are added, is vortexed and mixes 30 seconds；
9) it is transferred on magnetic frame, stands 3 minutes absorption magnetic beads, inhale and abandon solution；
10) step 8) and 9) primary is repeated；
11) it is centrifuged, the drop on centrifugation tube wall described in collecting pipe is transferred on magnetic frame, inhales and abandon solution；
12) it is air-dried 3~5 minutes；
13) plus the eluent of 52 μ L, vortex break up magnetic bead, are placed at room temperature for 5~10 minutes, and it is molten during which to shake 2~3 acceleration DNA
Solution；
13) it is transferred on magnetic frame, stands 3 minutes, the DNA for shifting dissolution is saved into new centrifuge tube, the DNA of the dissolution
As free Circulating tumor DNA.
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